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© The Rockefeller University Press, 0021-9525/1999//801 $5.00
The Journal of Cell Biology, Volume 146, Number 4, , 1999 801-818

Domains in the 1 Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas

^a Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455
^b Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, Colorado 80309-0347
Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Box 206, 420 Delaware St. SE, 4-102 Owre Hall, Minneapolis, MN 55455.(612) 624-8118(612) 626-1901

mary-p{at}biosci.cbs.umn.edu

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1 Dhc of the I1 inner dynein arm. The 1 Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH₂- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1 Dhcs were recovered. The truncated 1 Dhc fragments lacked the dynein motor domain, but still assembled with the 1β Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1 motor domain in the I1 structure and further revealed the role of the 1 Dhc in flagellar motility and phototactic behavior.

Key Words: motors • dynein • flagella • phototaxis • inner arm

© 1999 The Rockefeller University Press

THE movement of cilia and flagella is powered by axonemal dyneins, a family of mechanoenzymes that convert the energy derived from ATP binding and hydrolysis into the sliding of adjacent outer doublet microtubules (Mitchell 1994; Witman et al. 1994; Porter 1996). Axonemal dyneins can be separated into two groups, the outer dynein arms and the inner dynein arms, which have different functions in generating and propagating the flagellar waveforms. The outer dynein arms provide power to the flagellar beat, as Chlamydomonas mutants lacking the outer arms generate normal waveforms, but swim with a reduced beat frequency (Mitchell and Rosenbaum 1985; Brokaw and Kamiya 1987). In contrast, the inner arms are essential for normal motility, as mutants with inner arm defects have near normal beat frequencies, but display aberrant waveforms (Brokaw and Kamiya 1987).

The Chlamydomonas outer arm is one of the most well characterized dynein complexes; it is composed of three dynein heavy chains (Dhc)¹ (, β, and ), two intermediate chains (IC), and several light chains (LCs) (<20 kD), and repeats every 24 nm along the length of the axoneme (Witman et al. 1994). Sequence comparisons between outer arm Dhcs indicate that they share greatest similarity in the central and COOH-terminal regions (Mitchell and Brown 1994, Mitchell and Brown 1997; Wilkerson et al. 1994; Gibbons 1995). This portion of the Dhc is thought to form a globular head domain that interacts transiently with the microtubule doublet during the cross-bridge cycle. The more variable NH₂-terminal third of the Dhc is thought to form a flexible stem domain that extends to the base of the dynein arm and interacts with the isoform-specific IC and LC subunits (Sakakibara et al. 1993). The ICs are involved in the assembly of the outer arm complex (Mitchell and Rosenbaum 1986; Mitchell and Kang 1991) and its attachment to the outer doublet microtubules in an ATP-insensitive manner (S.M. King et al. 1991, S.M. King et al. 1995). The multiple LCs are thought to be involved in the regulation of motility, but their specific functions are largely unknown (Harrison and King 1999).

The inner dynein arms share an overall structural similarity to the outer arms but are significantly more complex in both composition and function. Ion exchange chromatography and SDS-PAGE procedures have identified at least eight distinct inner arm Dhcs that are associated with specific ICs and LCs into seven different molecular complexes: one two-headed isoform (I1) and six single-headed isoforms (I2 and I3) (Goodenough et al. 1987; Kagami and Kamiya 1992). These isoforms are arranged in complex groups along the length of the axoneme (Piperno et al. 1990; Piperno and Ramanis 1991; Muto et al. 1991; Mastronarde et al. 1992; Gardner et al., 1994; S.J. King et al. 1994), but the relationship between these different isoforms and the multiple Dhc genes (Porter et al. 1996, Porter et al. 1999) is almost completely unknown.

Very little is also understood about the mechanism by which any Dhc is targeted to its specific location within the axoneme. In this report, we focus on the role of the 1 Dhc in the assembly and targeting of the inner arm isoform known as the I1 complex. The I1 dynein provides several advantages for the study of dynein targeting. First, it is a relatively simple complex composed of two Dhcs (1 and 1β), three ICs of 140, 138, and 110 kD (Piperno et al. 1990; Smith and Sale 1991; Porter et al. 1992) and three LCs of 14, 12, and 8 kD (Harrison et al. 1998). Second, the I1 complex has a well-defined axoneme location, proximal to the first radial spoke in each 96-nm repeat along the length of the axoneme (Piperno et al. 1990; Mastronarde et al. 1992; Porter et al. 1992; Myster et al. 1997). Third, the I1 complex is an important target for the regulation of flagellar motility (Porter et al. 1992). Alterations in the phosphorylation state of IC138 have been associated with changes in microtubule sliding velocities and phototactic behavior (Habermacher and Sale 1997; King and Dutcher 1997). Finally, progress in the cloning and mapping of Dhc genes has identified two sequences that encode the I1 Dhcs (Porter et al. 1996; Myster et al. 1997; Perrone, C.A., R. Bower, S.H. Myster, J.A. Knott, and M.E. Porter, unpublished results). One of these sequences, Dhc1, maps to the PF9/IDA1 locus and encodes the 1 Dhc; mutations in this locus disrupt the assembly of the I1 complex and thereby alter flagellar motility (Myster et al. 1997). The availability of such mutations permits a functional analysis of the Dhc1 gene product in vivo.

To characterize the Dhc domains involved in the assembly and targeting of the I1 complex, we sequenced the complete Dhc1 transcription unit (>22 kb) and generated specific constructs of the Dhc1 gene. The constructs were used in cotransformation experiments to rescue the pf9 defects. These results represent the first full-length inner arm Dhc sequence to be described in any organism, and the first reported rescue of a Dhc mutation in Chlamydomonas. Our analysis of the Dhc1 transformants has also identified a subset of strains expressing truncated Dhc1 transcripts. The truncated transcripts encode NH₂-terminal fragments of the 1 Dhc polypeptide that are capable of coassembly with other components of the I1 complex and rebinding to the proper axoneme location. These results indicate that domains within the NH₂-terminal 143 kD of the 1 Dhc are involved in the specific subunit interactions required for the assembly and targeting of the I1 complex. EM analysis of isolated axonemes has identified the position of the 1 Dhc motor domain within the structure of the I1 complex. The assembly of truncated 1 Dhcs in the flagella of the transformants also resulted in a new motility phenotype that has revealed the contribution of the 1 Dhc motor domain to flagellar motility and phototactic behavior. These findings have important implications for the regulatory mechanisms that control the activity of the I1 dynein motor.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Origin of Genomic Clones and Sequence Analysis of the Dhc1 Gene
35 kb of genomic DNA in the region of the Dhc1 gene was recovered from a large insert, wild-type (21gr) Chlamydomonas library. The position of the Dhc1 transcription unit within this region was determined by probing Northern blots of wild-type RNA with selected subclones, and the Dhc1 transcription unit was thereby narrowed down to 22 kb of genomic DNA (see Fig. 1; Myster et al. 1997). Sequence information was obtained from both strands of subclones A–D, and G using a series of nested deletions (Erase-a-Base System; Promega Corp.) and Sequenase 2.0 (Amersham Life Science, Inc.) following the manufacturer's instructions. Subclones E and F were sequenced by the DNA Sequencing Facility (Iowa State University) on an ABI Prism sequencer (Perkin Elmer Corp.). The sequence data were assembled using the GCG software package versions 8 and 9 (Genetics Computer Group).

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Dhc1 gene structure. (a) Partial restriction map of the Dhc1 gene. The black box labeled P1 represents the 227 bp fragment of the Dhc1 gene that was recovered in the first PCR screen for Dhc genes in Chlamydomonas (Porter et al. 1996). This sequence was used to screen a large insert genomic library and recover >35 kb of genomic DNA surrounding the region encoding the hydrolytic ATP binding site (P1) (Porter et al. 1996; Myster et al. 1997). Indicated below are the approximate positions of selected SacI subclones (A–G) used as probes and other subclones (pSM8 and p14SE) that were used to construct a truncated Dhc1 gene. (b) Intron/exon structure of the Dhc1 transcription unit. The approximate size and position of the 29 exons (open boxes) and 28 introns (intervening lines) predicted from the analysis of the Dhc1 nucleotide sequence are shown. Also indicated are other features such as the TATA box, the regions encoding predicted P-loops, and a predicted polyadenylation signal at the 3' end of the gene. Bracketed regions numbered 1–5 indicate the position of the primers used for RT-PCR.

In five regions of the Dhc1 gene, the presence of multiple potential splice donor or acceptor sequences did not allow a confident prediction of the putative exons. In those cases, the splice junctions were determined directly by sequence analysis of reverse transcriptase–PCR (RT-PCR) products generated from the Dhc1 transcript (see Fig. 1). Total RNA was isolated from wild-type cells 45 min after deflagellation, and then 5 µg of total RNA was reverse transcribed using either a random primer or a sequence-specific reverse primer and the Superscript Preamplification System (GIBCO BRL) according to manufacturer's instructions. 5 µl of the resulting 25-µl cDNA product was used in a 100-µl PCR reaction with sequence specific primers. PCR reactions were performed using 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 2 mM deoxynucleotide triphosphates, 0.2 mM of each primer, and 2.5 U Taq polymerase (Life Technologies, Inc.). Some reactions also contained 3% DMSO. The PCR reactions were first denatured at 94°C for 3 min, followed by 30 cycles of 58°C for 1 min, 72°C for 3 min, and 94°C for 1 min, and then completed with a final cycle of 58°C for 1 min and 72°C for 5 min. The final reaction products were analyzed on agarose gels, and then purified using Wizard PCR preps (Promega Corp.) for direct sequencing with sequence-specific primers.

The proposed translation start site was determined by the recovery of an RT-PCR product using a forward primer downstream of the TATA box sequence and a reverse primer in exon 3. The resulting RT-PCR product contained stop codons in all three frames immediately preceding the proposed start codon.

The predicted amino acid sequence encoded by the Dhc1 gene was analyzed using the GCG program Motifs. The programs Bestfit, Compare, and Pileup were used to compare the 1 Dhc sequence to Chlamydomonas outer arm Dhc sequences , β, and (Mitchell and Brown 1994, Mitchell and Brown 1997; Wilkerson et al. 1994) and the cytoplasmic Dhc from Dictyostelium (Koonce et al. 1992). Regions with the potential to form -helical coiled coils were identified using the program COILS, version 2.2 (Lupus et al. 1991; Lupus 1996).

Cosmid Library Screening and Construction of pD1SA
To identify clones that might contain a full-length Dhc1 gene, we screened two different Chlamydomonas cosmid libraries (Purton and Rochaix 1994; H. Zhang et al. 1994) that were generously provided by S. Purton (University College, London) and D. Weeks (University of Nebraska). Because the Purton library contains the ARG7 gene within the cloning vector, the cosmid clones can be used to directly transform arg7 strains. 10⁵ independent clones from each library were screened on Magnagraph (Micron Separations, Inc.) nylon membrane lifts in duplicate with probes from the 5' and 3' ends of the Dhc1 gene (see Fig. 5 A). Probes used for hybridization were purified in low melting point agarose (GIBCO BRL) and radiolabeled with [³²P]dCTP and random hexamer primers using the Prime It II kit (Stratagene). Conditions for prehybridization and hybridization were as described previously (Porter et al. 1996; Porter et al. 1999; Myster et al. 1997). After single colony isolation, cosmid DNA was purified using alkaline lysis procedures and CsCl gradient centrifugation (Sambrook et al. 1989).

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Constructs of the Dhc1 gene used in the cotransformation experiments. (A) Partial restriction map of the Dhc1 gene. The arrow above identifies the position of the Dhc1 transcription unit within this region (Myster et al. 1997). SacI subclones (black and gray boxes) were used to screen two cosmid libraries to identify clones that contain the complete Dhc1 transcription unit. (B) Partial restriction map of the cosmid cA1. The approximate locations of the regions encoding the primary ATP hydrolytic site (P1) and the peptide (peptide) recognized by the 1 Dhc specific antibody (Myster et al. 1997) are shown. The cA1 clone is incomplete on the 3' end of the Dhc1 gene; it encodes up to residue 4,105 of the 1 Dhc sequence followed by the addition of 87 novel amino acids. Before transformation, the cA1 cosmid was linearized with BspE1, leaving >8 kb of flanking genomic DNA upstream of the predicted translation start site. The ARG7 sequence used as a selectable marker is located downstream of the Dhc1 sequence. (C) Partial restriction map of the cosmid cW1 containing a full-length Dhc1 gene. The regions encoding P1 and the peptide epitope are indicated, as are the predicted translation stop site (stop), and the consensus polyadenylation signal TGTAA (poly A signal). Before transformation, the cW1 cosmid was digested with PvuI, which releases the Dhc1 transcription unit as a 26-kb fragment flanked by 1.6 kb of genomic DNA on the 5' end and 4 kb of genomic and vector DNA on the 3' end. (D) Construction of a truncated version of the Dhc1 gene. An 11-kb SalI-AscI fragment from pSM8 was subcloned into the SalI-AscI digested p14SE plasmid. The resulting pD1SA construct contains 1.7 kb of genomic DNA 5' of the predicted translation start site, the region encoding the first 1,956 amino acids, including the epitope recognized by the 1 Dhc antibody, and an 1-kb fragment containing the 3' end of the gene. The shift in the open reading frame after the AscI site results in the addition of nine novel amino acids (QCHGCGPGV) followed by a stop. This construct was linearized with SalI before transformation. (e) Recovery of BAC clones containing the Dhc1 gene. Two different, large insert BAC clones were used in cotransformation experiments. The insert in the N24-1 clone contains 18 kb upstream and 52 kb downstream of the Dhc1 gene, whereas the insert in J1-5 contains 52 kb of genomic DNA both upstream and downstream of the Dhc1 gene. These constructs were not linearized before transformation.

Recovery of Bacterial Artificial Chromosome (BAC) Clones Containing the Dhc1 Gene
A modified pBELO BAC library containing Chlamydomonas genomic DNA was screened with selected subclones to identify large insert BAC clones containing the Dhc1 gene. This library was constructed by N. Haas and P. Lefebvre (University of Minnesota, St. Paul, MN) using genomic DNA from the cell-wall less strain cw92 and is currently available from Genome Systems, Inc. BAC DNA was isolated from positive clones using a modified version of the manufacturer's protocol available from C. Amundsen (University of Minnesota, St. Paul, MN) at the following URL: http://biosci.cbs.umn.edu/~amundsen/chlamy/methods/bac.html. The final pellet of BAC DNA was resuspended in 200 µl of TE and stored at –20°C. To identify clones containing full-length Dhc1 genes, 5 µl of BAC DNA was digested with the restriction enzyme SacI and analyzed on Southern blots using subclones from the 5' and 3' ends of the Dhc1 gene.

Nucleic Acid Analysis
Large-scale preparations of genomic DNA were isolated from wild-type and mutant transformant strains using CsCl gradients as described in Porter et al. 1996. A smaller scale mini-prep procedure (Newman et al. 1990) was used to isolate DNA samples from tetrad progeny and some of the transformants. Restriction enzyme digests, agarose gels, isolation of total RNA, and Southern and Northern blots were performed as previously described (Porter et al. 1996, Porter et al. 1999; Myster et al. 1997).

Cell Culture, Mutant Strains, and Cotransformation Experiments
The strains used in this study are listed in Table . All cells were maintained as vegetatively growing cultures at 21°C as previously described (Myster et al. 1997). The arg2 (Eversole 1956) strains were grown on rich medium that contained reduced ammonium nitrate (one tenth the normal concentration), but was supplemented with L-arginine to 0.6 mg/ml.

Analysis of Motility
Positive transformants were picked into 96-well plates and screened for motility on an inverted microscope (Olympus CK). Wells containing motile cells were streaked for single colonies and rescored on a phase-contrast microscope (Axioskop; Carl Zeiss, Inc.) using a 40x objective and a 10x eyepiece. The phenotypes of motile transformants were further analyzed by measuring forward swimming velocities and beat frequencies as previously described (Porter et al. 1992, Porter et al. 1994; Myster et al. 1997).

Transformants were tested for their ability to phototax using two different assays. In the first assay (King and Dutcher 1997), actively swimming cells were put in a dark box with a 3-cm-wide horizontal slit cut out along the bottom such that only the lower portion of a 10-ml suspension was illuminated. The box was placed 35 cm from a fluorescent light source for 40 min. Positively phototactic cells would concentrate in the lower, illuminated portion of the tube, whereas phototaxis defective cells would remain uniformly suspended throughout the tube. In the second assay, motile cells were transferred to a 96-well plate and placed on a dissecting microscope with substage illumination. Thick posterboard was placed under the plate and positioned to cover half of the well. The location of the cells within the well was followed over a 60-min time course. Positively phototactic cells would move quickly to the side of the well exposed to light, whereas cells unable to phototax would remain uniformly distributed throughout the well.

To verify that the rescued motility in the transformants was due to expression of the Dhc1 transgene and not a reversion event at the PF9 locus, the motile transformants were backcrossed to a pf28 allele (oda2), which lacks the outer arms but is wild-type at the PF9 locus (Kamiya 1988). Tetrad progeny were recovered following standard genetic methods (Harris 1989), and their motility phenotypes were scored using a phase-contrast microscope as described above.

Axoneme Isolation, Dynein Extracts, and Sucrose Gradients
Axonemes were prepared from large-scale (5–40 liters) liquid cultures of vegetative cells using procedures described by Witman 1986 and S.M. King et al. 1986, as modified by Gardner et al. (1994) and Myster et al. 1997. Crude dynein extracts were obtained by brief (30 min) high salt extraction of whole axonemes (Porter et al. 1992; Myster et al. 1997). To isolate I1 complexes, crude dynein extracts were fractionated by sucrose density gradient centrifugation as previously described (Porter et al. 1992; Myster et al. 1997). Aliquots of each fraction were analyzed by SDS-PAGE and Western blotting.

SDS-PAGE and Immunoblot Analysis
Protein samples from whole axonemes and sucrose gradient fractions were separated on 5% polyacrylamide gels using the Laemmli 1970 buffer system and either stained directly with Coomassie brilliant blue (R250; Sigma Chemical Co.) or transferred to either nitrocellulose (Schleicher and Schull, Keene) or Immobilon-P (Millipore) in 25 mM Tris, 192 mM glycine, and 12.5% methanol at 800 mA for 90 min at 4°C using a Genie electroblotter (Idea Scientific, Co.). Nitrocellulose blots were blocked in 1x PBS (0.58 M Na₂HPO₄, 0.017 M NaH₂PO₄·H₂O, 0.68 M NaCl), 5% normal goat serum (Sigma Chemical Co.), and 0.05% Tween 20 (polyethylenesorbitan monolaurate), whereas the Immobilon-P membrane was blocked in 0.2% I-Block (Tropix) in 1x PBS and 0.5% Tween 20.

Four different antibody preparations were used to probe the blots. The 1 Dhc antibody has been previously described in detail and is highly specific for the Dhc1 gene product (Myster et al. 1997). This antibody was raised against the peptide sequence DGTCVETPEQRGATD, which corresponds to amino acids 1,059–1,073 of the 1 Dhc polypeptide. The 1 Dhc antibody was affinity-purified on Western blots of dynein extracts, and then used at a 1:10 dilution. The IC140 antibody was provided by P. Yang and W. Sale (Emory University, Atlanta, GA). This antiserum was raised against a fusion protein containing a fragment of the 140-kD intermediate chain of the I1 complex (Yang and Sale, 1998), and it was typically used at a dilution of 1:3,000. The rabbit polyclonal antibody R5205, which was raised against a fusion protein of the human 14-kD dynein LC (S.M. King et al. 1996), was provided by S. King (University of Connecticut). The R5205 antibody cross-reacts with the 14-kD LC (Tctex1) of the I1 complex (Harrison et al. 1998) and was used at a dilution of 1:50. After incubation overnight at 4°C, the blots were washed in 1x PBS and 0.05% Tween 20. Immunoreactivity was detected using an alkaline phosphatase–conjugated secondary antibody, BCIP (5-bromo-4-chloro-3-indolyl phosphate), and NBT (nitro blue tetrazolium) following the manufacturer's instructions (Sigma Chemical Co.). An mAb to tubulin (T5168; Sigma Chemical Co.) was used at a dilution of 1:1,000, and then detected using an HRP-conjugated secondary antibody, 4-chloro-1-naphthol, and hydrogen peroxide following the manufacturer's protocol (Sigma Chemical Co.).

Electron Microscopy and Image Analysis
To view the I1 complex in strains with rescued motility, selected transformants were crossed to a pf9-3 strain to recover strains with rescued I1 complexes and the wild-type complement of outer dynein arms. Axonemes were prepared and processed for EM as previously described (Porter et al. 1992; Myster et al. 1997). Longitudinal images were selected, digitized, and averaged using the methods described in Mastronarde et al. 1992. Averages of individual axonemes were obtained by analyzing at least six 96-nm radial spoke repeats, and then averages from several axonemes were combined to obtain a grand average for each strain. The methods used to compute differences between two strains are described in detail in Mastronarde et al. 1992.

Recovery of Dhc1 Transgene from G3 After Transformation
To identify the 3' end of the Dhc1 transgene in the G3 transformant (which assembles the shortest 1 Dhc fragment), genomic DNA was isolated from wild-type and G3, digested with the restriction enzymes SacI and KpnI, and analyzed on Southern blots probed with Dhc1 subclones. A polymorphic 7.2-kb SacI-KpnI fragment was identified in G3 using subclone C. This polymorphic fragment was recovered from G3 genomic DNA by constructing a size-selected minilibrary, and then screening the library with subclone C. After single colony purification, the 3' end of the truncated Dhc1 transgene was sequenced with Dhc1 specific primers to determine the predicted amino acid sequence at the COOH terminus of the 1 Dhc fragment.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Sequence Analysis of the Dhc1 Transcription Unit
In previous work, we identified a null mutation in the Dhc1 gene that resulted in the failure to assemble the I1 inner arm complex into the flagellar axoneme (Myster et al. 1997). To understand how the Dhc1 gene product might contribute to dynein complex formation, we have now sequenced the entire Dhc1 transcription unit (Fig. 1 a). A map of the deduced gene structure is shown in Fig. 1 b. Sequence analysis of subclone A identified the 3' end of the neighboring gene (geranyl geranyl pyrophosphate synthase), 800 bp of intervening sequence, and a TATA box sequence 144 bp upstream of the proposed translation start site of the Dhc1 gene (see Materials and Methods). All of the 5' sequence elements required for regulated Dhc1 expression should therefore be contained within an 1-kb region. The next 20 kb of the sequence contains the coding region located within 29 exons. The 3' end of the gene is located in subclone G, which contains the last exon encoding the COOH-terminal 552 amino acids, a stop codon, and a consensus polyadenlyation signal sequence (TGTAA) 471 bp downstream.

The predicted amino acid sequence of the encoded 1 Dhc contains 4,625 amino acid residues and corresponds to a polypeptide of 522,806 D (Fig. 2). A search for potential nucleotide binding sites within the 1 Dhc sequence identified six consensus or near consensus phosphate-binding (P-loop) motifs with the sequence A/GXXXXGKT/S (Walker et al. 1982). Four of the P-loop motifs (P1–P4) are located within the central region of the Dhc, and both spacing and sequences of these P-loops are similar among all Dhc sequences reported thus far (reviewed in Gibbons 1995; Porter 1996). Two additional P-loop motifs were identified in the NH₂-terminal (Pn) and COOH-terminal (Pc) regions of the 1 Dhc respectively; these appear to be unique to the 1 Dhc (Fig. 2).

View larger version (75K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Predicted amino acid sequence of the 1 Dhc. Sequences identified as P-loop motifs are indicated by a single underline. The amino acid sequence of the peptide used for antibody production by Myster et al. 1997 is indicated by asterisks. The arginine (R) residue at the COOH-terminal end of the truncated 1 Dhc in the G3 transformant is indicated by bold and an underline. The complete nucleotide sequence is available from GenBank/EMBL/DDBJ under accession number AJ243806.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Comparisons between Dhc sequences. The 1 Dhc was compared with the Chlamydomonas outer arm Dhc sequences (, β, and ) and the Dictyostelium cytoplasmic Dhc using the GCG program COMPARE with a window of 50 residues and a stringency of 22. The accession numbers for these sequences are as follows: L26049, U02963, U15303, and Z15124.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Secondary structure of the 1 Dhc. Shown here is a graphical representation of the regions of the 1 Dhc predicted to form -helical coiled coils, as determined by the program COILS version 2.2 (Lupus et al. 1991; Lupus 1996). The six regions of the sequence predicted to encode P-loops are identified by arrows. The position of the peptide sequence (amino acids 1,059-1,073) used to generate an isoform specific antibody (Myster et al. 1997) is also indicated.

Rescue of pf9 Motility Defects by Transformation with Constructs of the Dhc1 Gene
Mutations at the PF9/IDA1 locus typically result in strains that have a slow, smooth swimming behavior (Kamiya et al. 1991; Porter et al. 1992). To increase the sensitivity of the screen for rescue of the pf9 mutant phenotype, we introduced a second motility mutation into the pf9-2 background. pf28 is a mutation in the Dhc gene that results in the failure to assemble the outer dynein arms (Mitchell and Rosenbaum 1985; Wilkerson et al. 1994). Cells carrying the pf28 mutation swim with a jerky phenotype that can be easily distinguished from the slow, smooth swimming behavior of the pf9 mutant cells. In addition, pf9-2 pf28 double mutants assemble short, paralyzed flagella and sink in liquid medium (Porter et al. 1992). This short, paralyzed flagellar phenotype makes it very straightforward to identify transformants that have rescued the pf9 mutant defects by screening for cells that assemble full-length, motile flagella and swim with a pf28-like motility phenotype.

The pf9-2 pf28 arg2 strain was first cotransformed with the selectable marker pARG7.8 and the cW1 cosmid containing the complete Dhc1 transcription unit. Positive transformants were selected by growth on solid medium lacking arginine, and then single colonies were picked into liquid media and screened for motility. Fig. 5 C illustrates the combined results of several independent cotransformation experiments with the cW1 cosmid. Although arg+ transformants were recovered at expected frequencies (Kindle 1990), only 11 out of 2,880 transformants screened displayed any motility, for a frequency of rescue of <0.4%. Moreover, the motility of the cW1 rescued strains was not the same as pf28 (see below and Table ).

Previous study of an outer arm mutation, oda4-s7, had indicated that the NH₂-terminal third of the β Dhc polypeptide is sufficient for assembly of a dynein complex (Sakakibara et al. 1993). To test if this is also true for the 1 Dhc, we cotransformed the pf9 pf28 mutant with the smaller pD1SA construct, which encodes 40% of the 1 Dhc sequence. These experiments yielded seven motile strains (Fig. 5 D), which represented only a modest (0.87%) increase in the frequency of rescue, but these rescues confirmed that truncated Dhc1 transgenes could restore partial motility. To see if it was possible to completely rescue the motility defects, we also transformed the pf9 pf28 mutant with two BAC clones that contained the full-length Dhc1 gene located in the middle of 100–135-kb genomic inserts (Fig. 5 E). The frequency of rescue (0.1%) was still quite low, but the motility phenotypes of the rescued strains were very similar to pf28 (see below).

The recovery of motile isolates after cotransformation could also be due to an intragenic reversion event at the PF9 locus during the course of transformation and/or selection. To confirm that the motility of the transformants was due to the successful expression of the Dhc1 transgene and not a reversion event, two of the cW1 transformants (E2 and G4) were crossed with oda2, another mutant allele at the PF28/ODA2 locus, and the progeny from 11 complete tetrads were analyzed for each cross. If the rescued motility was due to reversion of the pf9-2 mutation, all the resulting tetrad progeny would be motile and swim with a pf28/oda2–like motility phenotype. However, if the rescued motility was due to the presence of the Dhc1 transgene, then the restored motility phenotype would be expected to segregate independently of the pf9-2 mutation, and a class of immotile progeny with the original pf9-2 pf28 genotype should be recovered. Surprisingly, we observed three different motility phenotypes in the tetrad progeny. The first class swam with a motility phenotype that was indistinguishable from either pf28 or oda2. The second class swam with a jerky motion like the pf28/oda2 strains but appeared slower. The third class was immotile with short, stumpy flagella. The recovery of aflagellate strains demonstrated that the original pf9-2 mutation was still present in the genetic background of the two transformants and that the rescued motility was due to the presence of the Dhc1 transgene.

Motility Phenotypes of the Dhc1 Transformants
Although the frequency of rescue was low, it was clear that the rescued motility was due to the presence of the different Dhc1 transgenes, and so the motility phenotypes of the Dhc1 transformants were analyzed in greater detail. More specifically, we measured the flagellar beat frequency, the forward swimming velocity, and the ability to phototax (Table ). Transformants with complete rescue of the pf9 mutation would be expected to have a swimming phenotype nearly identical to that of pf28. The flagellar beat frequencies of the Dhc1 transformants were almost identical to the beat frequency of pf28, but measurements of forward swimming velocities clearly indicated that most of the transformants swam more slowly than pf28 (Table ). In particular, the swimming velocities of the rescued strains obtained by transformation with the cosmid clones and pD1SA were slower than those obtained by transformation with the BAC clones. These results suggested that there were still some inner arm defects in most of the Dhc1 transformants.

We next tested if the Dhc1 transformants had recovered the ability to phototax. King and Dutcher 1997 have previously used a photoaccumulation assay to demonstrate that pf9 mutant cells do not phototax effectively. Using similar conditions, we have found that pf28 cells, which lack outer arms but have the full complement of inner dynein arms, are able to phototax, as assayed by their tendency to become concentrated in the illuminated portion of a tube within 40 min of exposure to a directional light source (Table ). However, all of the Dhc1 transformants obtained with the cosmid clones remained equally distributed between the illuminated and darkened regions of the tube. To confirm these findings by direct observation of individual cells, the ability to phototax was also monitored in 96-well plates over a 60-min time course (see Materials and Methods). In the absence of outer arms, the Dhc1 transformants obtained with the cosmid clones remained equally distributed in both the illuminated and darkened portions of the microtiter well. Conversely, the majority of pf28 cells became concentrated on the illuminated side of the well within 15 min. These results indicated that this group of Dhc1 transformants does not phototax as effectively as pf28 control cells.

To examine the motility of the transformants in the presence of outer arms, two strains, G4 and E2, were crossed to pf9-3 and tetrad products containing the Dhc1 transgene in a wild-type outer arm background (G4+OA and E2+OA) were recovered. The two strains have beat frequencies almost identical to wild type, but their swimming velocities are intermediate in speed between pf9 and wild type (Table ). Moreover, in the presence of the outer arms, the two strains could photoaccumulate as effectively as wild type. These results suggest that the outer arms can compensate in some way for the phototaxis defects in the Dhc1 transformants.

Truncated 1 Dhcs in the Dhc1 Transformants
The swimming behavior of the Dhc1 transformants obtained with the cosmid clones demonstrated that the introduction of these Dhc1 clones resulted in only a partial rescue of the pf9 motility defects. Given the large size of the Dhc1 transcription unit (>22 kb), we were initially concerned that these transgenes might not be expressing wild-type levels of the Dhc1 gene product. To address this question, we isolated axonemes from the Dhc1 transformants and analyzed the components of the I1 complex. Previous work has shown that the I1 complex is composed of eight polypeptides, two Dhcs (1 and 1β), three ICs (IC140, IC138, and IC110) (Smith and Sale 1991; Porter et al. 1992; Myster et al. 1997), and three LCs (LC8, LC12, and LC14) (Harrison et al. 1998). The Dhc1 gene encodes the 1 Dhc, which can be identified on Western blots using antibody directed against a peptide epitope in the NH₂-terminal region (Myster et al. 1997). Fig. 6 A shows three Coomassie blue–stained polyacrylamide gels containing whole axonemes isolated from several mutant strains: the 11 motile Dhc1 transformants obtained with the cW1 cosmid, the 4 motile Dhc1 transformants recovered with the cA1 construct, and the 5 transformants recovered with the Dhc1 BAC clones. Fig. 6 B shows the corresponding Western blots probed with the 1 Dhc antibody. The 1 Dhc antibody identified the 520 kD 1 Dhc in the pf28 control sample, but polypeptides significantly smaller than the 1 Dhc were identified in all of the motile strains obtained by transformation with the Dhc1 cosmids. In contrast, all of the axoneme samples prepared from rescued strains obtained by transformation with the Dhc1 BAC clones contained full-length 1 Dhc polypeptides. These results indicated that the partial rescue phenotype seen with the Dhc1 cosmid clones was not due to low levels of expression of a full-length 1 Dhc, but instead due to the expression of truncated 1 Dhcs, ranging in size from 165 to 300 kD.

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Western blots of isolated axonemes from Dhc1 transformants. Isolated axonemes were prepared from the Dhc1 transformants, split into triplicate for separation on 5% polyacrylamide gels, and then either stained with Coomassie blue or transferred to membranes and incubated with the affinity-purified 1 Dhc antisera or the IC140 antisera. (A, left panel) A 5% polyacrylamide gel loaded with 20-µg whole axonemes from pf28, pf9 pf28, and the 11 motile Dhc1 transformants generated by transformation with the cW1 cosmid. The additional bands in the pf9 pf28 sample are most likely contaminating flagellar membrane proteins. (Middle panel) A 5% gel loaded with whole axonemes from pf28, pf9-3, and the four rescued strains generated by transformation with the cA1 cosmid. (Right panel) A 5% gel loaded with whole axonemes from pf28 and the five rescued strains obtained with the BAC clones. (B) Duplicate immunoblots probed with the affinity-purified 1 Dhc antibody. (C) Duplicate immunoblots probed with the IC140 antisera. Control blots probed with tubulin antibodies confirmed that roughly equivalent amounts of flagellar protein were loaded in each lane (data not shown).

Assembly of I1 Complexes in Dhc1 Transformants
To confirm that the other polypeptide subunits were assembled into an I1 complex, we isolated whole axonemes from large-scale cultures of two Dhc1 transformants, E2 and G4, as well as from control pf28 cells. Partially purified I1 complexes were obtained by high salt extraction of the isolated axonemes followed by sucrose density gradient centrifugation. The resulting fractions were analyzed by both SDS-PAGE and Western blotting. Fig. 7 A shows the 19S region of a sucrose gradient that was loaded with the pf28 dynein extract. The two Dhcs and three ICs of the I1 isoform cosediment as a complex that peaks in fraction number 4. Duplicate samples tested on Western blots probed with the 1 Dhc antibody confirmed the presence of the 1 Dhc in the 19S region (right). Fig. 7 B shows four fractions from the sucrose gradient that was loaded with the G4 dynein extract. The gel on the left reveals that the 1β Dhc and the three intermediate chains of the I1 complex have shifted and now cosediment at 16S, peaking in fraction 6. A novel polypeptide of 183 kD cosediments in the same region (see asterisks). Western blot analysis with the 1 Dhc antibody identified this novel band as the truncated 1 Dhc (Fig. 7 B, blot). Identical results were observed with dynein extracts isolated from the E2 strain (data not shown). The truncated 1 Dhcs in the Dhc1 transformants therefore form stable complexes with the other polypeptides of the I1 complex, but the resulting mutant complexes sediment more slowly than wild-type complexes.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Cosedimentation of the truncated 1 Dhc with other subunits of the I1 complex. Whole axonemes were isolated from pf28 and the Dhc1 transformant G4 and extracted with high salt to release the dynein arms. The crude dynein extracts were loaded onto a 5–20% sucrose gradient and centrifuged for 15 h at 33,500 g. Duplicate samples from the 19S region of the gradients were separated on 5% polyacrylamide gels and either stained with Coomassie blue or transferred to nitrocellulose and incubated with the affinity-purified 1 Dhc antibody. (A) Gel and corresponding immunoblot from the pf28 extract. (B) Gel and corresponding immunoblot from the G4 extract. The asterisk identifies the truncated 1 Dhc that cosediments at 16S with the other subunits of the I1 complex.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Structural defects in Dhc1 transformants. Analysis of longitudinal images of axonemes from (a) wild-type, (c) pf9-3, (e) G4+OA, and (g) E2+OA. Grand averages for wild-type, pf9-3, G4+OA, and E2+OA based on 9, 6, 10, and 9 individual axonemes and 62, 44, 89, and 77 repeating units, respectively. (b) Outline of the individual densities in the 96-nm axonemal repeat. OA indicates positions of four outer dynein arm complexes per 96-nm repeat. S1 and S2 indicate the proximal and distal radial spokes, respectively. IA indicates the structures (numbered 1–10) seen in the inner arm region. (d) Difference plot between wild-type and pf9-3, with differences not significant at the 0.005 confidence level set to zero. (f) Difference plot between wild-type and G4+OA. (h) Difference plot between wild-type and E2+OA.

Fig. 9 A shows a partial restriction enzyme map of the Dhc1 gene and the subclones that were used as probes to analyze the Dhc1 transcripts. As shown in Fig. 9 B, probe A3', which spans the Dhc1 transcription start site, identified a single, large (>13 kb) transcript in wild-type RNA. However, in G4+OA and E2+OA, the transcripts recognized by the A3' probe were significantly smaller than the wild-type Dhc1 transcript, but these smaller transcripts were still upregulated in response to deflagellation (compare lanes 0 and 45). Identical results were observed with the next two subclones, probes B and C. Probe D, which includes the conserved region encoding the primary ATP hydrolytic site, hybridized to the truncated transcripts in E2+OA, but it did not recognize the truncated transcripts in G4+OA. Probes E–G did not hybridize with any transcripts in the transformants (Fig. 9 B, right panel, and data not shown). The Dhc1 transcripts in G4 and E2 are therefore truncated from the 3' end of the Dhc1 gene, and G4 is truncated before E2.

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 9 Northern blot analysis of Dhc1 transcripts in rescued strains. (A) Partial restriction map of the Dhc1 gene and subclones used as probes in the Northern analysis. (B) Northern blots of total RNA from wild-type, G4+OA, and E2+OA, isolated before (0) and 45 min (45) after deflagellation. Parallel samples of 20 µg total RNA were separated on 0.75% formaldehyde-agarose gels, transferred to Zetabind, and hybridized with selected Dhc1 subclones. Each probe hybridized to a single, large (>14 kb) transcript in wild-type (wt). Probe A3' (left panel) also hybridized to truncated Dhc1 transcripts that are upregulated in response to deflagellation in G4+OA and E2+OA. Probes B and C gave similar results. Probe D (middle panel) hybridized to the truncated Dhc1 transcript in E2+OA, but not in G4+OA. (Probe D, which corresponds to the most highly conserved region of the Dhc1 gene, also cross-hybridized weakly with other Dhc transcripts in these samples.) Probe G (right panel) did not detect any transcripts in either G4+OA or E2+OA, and similar results were seen with probes E and F. (C) Northern blots of total RNA from wild-type, G3, A2, and G9 isolated 45 min (45) after deflagellation. Each probe hybridized to a single transcript in wild-type (wt) as well as the endogenous Dhc1 transcript present in the pf9-2 background of the transformants (Myster et al. 1997). Probe C (left panel) hybridized to the truncated Dhc1 transcripts in G3, A2, and G9, but probes D–F (middle panel) did not.

Recovery of a Modified Transgene from a Dhc1 Transformant
To identify the sites where the Dhc1 cosmid clones were being modified during transformation, we isolated genomic DNA from the transformants and analyzed the structure of the integrated Dhc1 transgenes on Southern blots. Fig. 10 A shows a blot of SacI digested genomic DNA that was hybridized with a probe for subclone C. As expected, this probe hybridized to the endogenous Dhc1 gene present in the pf9-2 mutant background of the transformants. However, for each transformant, a second polymorphic band could be detected using either probe C (Fig. 10 A) or probe D (data not shown). From these and other blots, we concluded that the Dhc1 cosmids were being rearranged during integration into genomic DNA. In addition, the region of the Dhc1 gene encoding the conserved motor domain appeared to be the most common target for disruption during these integration events.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Modification of the Dhc1 transgenes in the transformants. (A) Southern blot analysis of the Dhc1 transgenes after integration into the pf9-2 pf28 host strain. DNA was isolated from pf9-3, pf28, pf9-2, the H10 strain obtained by transformation with pD1SA, and the 11 rescued strains generated by transformation with cW1. 4 µg of genomic DNA was digested with SacI, separated on an 0.8% agarose gel, transferred to Magnagraph, and hybridized with probe C (Fig. 1). Probe C identifies the endogenous 4.2-kb SacI fragment in every sample except pf9-3, which contains a 13-kb deletion of the Dhc1 gene (Myster et al. 1997). Probe C also hybridizes to novel restriction fragments in a subset of the transformants, which indicates that this region was a frequent site of rearrangement of the Dhc1 transgenes. Rearrangements in the region corresponding to probe D were identified in the remaining samples (data not shown). (B) Determination of the predicted amino acid sequence of the 1 Dhc fragment in the G3 transformant. The truncated Dhc1 transgene was recovered by screening a mini-library constructed from G3 genomic DNA (see Materials and Methods). The 3' end of the transgene was sequenced with Dhc1 specific primers. The nucleotide sequence revealed that the Dhc1 transgene was fused to an unidentified DNA sequence. The resulting hybrid gene encodes up to residue 1,249 of the 1 Dhc, and then adds 17 novel amino acids before encountering a stop. (C) Schematic representation of the truncated 1 Dhc polypeptide present in transformant G3. The top line is the wild-type 1 Dhc showing the approximate positions of the P-loops, the predicted coiled-coil domains, and the peptide epitope recognized by the 1 Dhc antibody. Below is a similar diagram of the 1 Dhc fragment in G3. The sequence ends at amino acid 1,249, shortly after the epitope recognized by the 1 Dhc antibody.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
16 different Dhc genes have been identified in Chlamydomonas: 2 cytoplasmic Dhc sequences (Pazour et al. 1999; Porter et al. 1999), 3 genes that encode the Dhcs of the outer dynein arm (Mitchell and Brown 1994, Mitchell and Brown 1997; Wilkerson et al. 1994), and 11 other Dhc genes whose expression patterns are consistent with gene products that are involved in flagellar function (Porter et al. 1996, Porter et al. 1999; Knott, J., and M. Porter, unpublished results). The analysis of specific Dhc mutations has revealed the role of each outer arm Dhc in flagellar motility (Sakakibara et al. 1991, Sakakibara et al. 1993; Porter et al. 1994; Rupp et al. 1996), but the specific functions of the multiple inner arm Dhcs are largely unknown. We have previously demonstrated that the Dhc1 gene product plays an essential role in the assembly and function of the I1 inner arm complex (Porter et al. 1996; Myster et al. 1997). To better understand how this inner arm Dhc contributes to flagellar motility, we have now analyzed the structure and function of the Dhc1 gene and the encoded 1 Dhc in detail.

Sequence Analysis of Dhc1 Transcription Unit
As described in Fig. 1, we have determined the nucleotide sequence for the complete Dhc1 transcription unit and used this information to obtain the predicted amino acid sequence of the 1 Dhc (Fig. 2). To our knowledge, this is the first full-length, inner arm Dhc sequence to be reported in any organism. Comparisons to other full-length Dhc sequences indicate that the 1 Dhc is most similar to the β and Dhcs of the outer arm, and that these sequence similarities extend into the NH₂-terminal region (Fig. 3). As the NH₂-terminal region of the Dhc is thought to be involved in the association with isoform specific IC and LC subunits, these observations suggest that the 1 Dhc and the β and Dhc contain conserved sites for the binding of accessory subunits. Indeed, recent studies have revealed that the I1 complex and the outer arm do contain similar IC and LC components, such as the 8-kD LC, the Tctex1 and Tctex2 LCs, and a family of WD-repeat containing ICs (Patel-King et al. 1997; Harrison et al. 1998; Yang and Sale, 1998). However, a novel feature of the 1 Dhc sequence is the presence of a P-loop motif (Pn) at amino acid residues 960–967 (Fig. 2). A weakly conserved P-loop motif has also been identified within the first 200 residues of the β Dhc (Mitchell and Brown 1994; Kandl et al. 1995). Whether the Pn sequence in the 1 Dhc is a bona fide nucleotide binding site is unknown, but the future sequence analyses of 1 Dhc homologues in other organisms should indicate whether the Pn motif is a conserved feature of this class of Dhc.

The central and COOH-terminal thirds are the most highly conserved regions of the 1 Dhc, and our transformation experiments are consistent with previous proposals that this region corresponds to the dynein motor domain (Sakakibara et al. 1993; Koonce and Samso 1996, Gee et al. 1997). The amino acid residues around P1 (the primary ATP hydrolytic site) and P4 are highly conserved with other axonemal Dhcs, whereas the sequences around P2 and P3 are less well conserved (Gibbons et al. 1994; Gibbons 1995). For example, P3 does not strictly conform to the P-loop consensus sequence GXXXXGKS/T (Walker et al. 1982), as the glycine in position 6 is substituted by an alanine in the 1 Dhc sequence, but the same amino acid substitution was found in the Dhc sequence (Wilkerson et al. 1994).

The COOH-terminal third of the 1 Dhc also contains a small region 340 amino acids downstream from P4 that is predicted to form a limited coiled-coil domain (Fig. 4). A similar region in cytoplasmic Dhc sequences has been identified recently as the stalk structure that extends from the globular head domain and forms the microtubule binding site (Koonce 1997; Gee et al. 1997; Gee and Vallee 1998). Mutations in this region in the outer arm β Dhc can have dramatic effects on flagellar motility (Porter et al. 1994). Interestingly, a sixth P-loop motif (Pc) has been identified in the 1 Dhc sequence, 280 amino acids downstream from the proposed microtubule binding site. The function of this sixth P-loop in the 1 Dhc is unknown, but its position downstream from the microtubule binding site is intriguing. Recent sequence analysis has suggested that all dyneins may contain six ATPase-like repeat regions: the four central P-loops previously identified and two additional, less well conserved repeats after the COOH-terminal coiled-coil domains (Neuwald et al. 1999).

Rescue of the pf9 Mutant Phenotype by Transformation with Dhc1 Constructs
Using the Dhc1 sequence information, we recovered two BAC clones and two cosmid clones containing full-length or near full-length Dhc1 genes, and then used these constructs to rescue the pf9 motility defects (Fig. 5). 20 independent transformants with rescued motility were recovered. Backcrossing the transformants confirmed that the rescued motility was due to the presence of the Dhc1 transgene and not to a reversion event at the PF9 locus. However, analysis of the Dhc1 transformants produced two unexpected results. First, the frequency of rescue (<1%) was much lower than previously observed with other flagellar genes (5–10%) (Diener et al. 1990; Kindle 1990; Tam and Lefebvre 1993), and second, the 1 Dhcs were truncated in most of the motile transformants recovered thus far.

One reason that the cotransformation frequencies were so low might be due to the large size of the Dhc1 transcription unit, which could make the Dhc1 transgenes more susceptible to damage during the transformation protocol. Exogenous DNA sequences often undergo deletions as they integrate into the Chlamydomonas genome (Tam and Lefebvre 1993; Smith and Lefebvre 1996, Smith and Lefebvre 1997; Koutoulis et al. 1997). The recovery of truncated 1 Dhc fragments (Fig. 6) indicated that the Dhc1 cosmids were being disrupted during the transformation protocol, and both Southern and Northern blot analyses of the rescued transformants (Fig. 9 and Fig. 10) have confirmed that deletions from the 3' end of the Dhc1 cosmids did occur. The presence of additional genomic DNA flanking the Dhc1 transcription unit in the BAC clones may have served to protect the Dhc1 transgenes and thereby permitted the full-length rescues observed with these clones (Fig. 5 and Fig. 6).

Another reason for the low frequency of rescue might be the relatively small amount of genomic DNA present on the 5' end of the Dhc1 gene in certain constructs (Fig. 5). If this region was randomly deleted, the resulting cotransformants would not retain the sequences necessary for expression of the Dhc1 transcript and rescue of the mutant phenotype. Recent experiments with constructs encoding the IC140 subunit have indicated that sufficient DNA upstream from the 5' end of the IC140 gene is essential for efficient rescue of the ida7 mutation (Perrone et al. 1998).

The NH₂-terminal 143-kD of the 1 Dhc Is Sufficient for Complex Assembly
The second unexpected result was the frequency with which we recovered motile transformants expressing only NH₂-terminal fragments of the 1 Dhc (Fig. 6 B). Northern and Southern blot analyses have shown that the rescued strains retained the 5' sequence elements required for regulated expression of the Dhc1 transcripts, but several of these strains lacked the 3' end of the transgene (Fig. 9 and Fig. 10). Therefore, the truncated 1 Dhcs represent those NH₂-terminal fragments that were competent to assemble with other subunits into the I1 complex (Fig. 6B and Fig. C, and Fig. 7).

The observation that none of the 1 Dhc fragments is smaller than 143 kD may indicate that this is the shortest NH₂-terminal fragment capable of complex assembly. Studies of outer arm mutants have identified a novel β Dhc mutation with similar properties. The oda4-s7 mutant expresses a 160-kD fragment of the β Dhc that is capable of coassembly with other outer arm subunits at the correct axoneme location (Sakakibara et al. 1993). Likewise, low level expression of cytoplasmic Dhc constructs in Dictyostelium indicates that a 158-kD NH₂-terminal fragment is also capable of complex assembly (Koonce and Knecht 1998).

Although the NH₂-terminal third of the 1 Dhc is the most variable region, secondary structure programs have identified a region just before P1 that is predicted to form a limited coiled-coil domain (Fig. 4). This domain, which has been identified in nearly all Dhcs sequenced to date (Mitchell and Brown 1994, Mitchell and Brown 1997), has been proposed as a potential region that might mediate interactions between the Dhcs and their associated ICs and LCs. To determine if this coiled-coil domain is required for assembly of the I1 complex, we recovered the Dhc1 transgene from the G3 transformant, which expresses the shortest 1 Dhc fragment (Fig. 6 B). Sequence analysis of the truncated Dhc1 transgene demonstrated that the 1 Dhc sequence terminates before the region predicted to form the NH₂-terminal coiled-coil domain (Fig. 10). Given that this 1 Dhc fragment still assembles with other I1 components into the flagellar axoneme (Fig. 6), other sites within the NH₂-terminal region must be required for complex formation. We plan to analyze additional Dhc1 constructs to further delineate the domains required for specific subunit interactions and complex assembly.

Assembly of the Dynein Motor Domain
If the Dhc1 transgenes were deleted randomly from the 3' end, we would expect to recover a broad distribution of Dhc fragments ranging in size from the minimum required to assemble the I1 complex to nearly full-length. Therefore, why are almost all of the 1 Dhc fragments smaller than 217 kD (Fig. 6 B)? One possibility may be that larger 1 Dhc fragments are unstable and prevent assembly of the I1 complex into the axoneme. Studies in Dictyostelium have shown that constructs of cytoplasmic Dhc lacking significant portions of the COOH terminus are expressed poorly as compared with other constructs that contain the entire motor domain (Koonce 1997). Alternatively, the presence of larger 1 Dhc fragments with partial motor domains may inhibit flagellar motility. If so, we would not recover such transformants in our screen, which was based on the rescue of a motility defect. Indeed, Northern blot analysis of the two transformants (A2 and G9) that assemble the largest 1 Dhc fragments demonstrates that the sequences encoding the dynein motor domain are not present in the associated Dhc1 transcripts (Fig. 9). The NH₂-terminal regions of these larger 1 Dhc fragments must, therefore, be fused to other protein sequences. Interestingly, the absence of motile transformants with partial motor domains is consistent with previous reports that nucleotide binding by the cytoplasmic Dhc is inhibited by deletion of the COOH terminus, leading to the formation of rigor complexes (Gee et al. 1997).

Implications for the Structure of the I1 Complex
Structural studies of the isolated I1 complex by negative staining or rotary shadowing have shown that it is a two-headed isoform (Goodenough et al. 1987; Smith and Sale 1991) but the physical relationship between the globular heads seen in vitro and the structural domains identified in situ has been unknown. Because some of the Dhc1 transformants assemble I1 complexes that lack central and COOH-terminal regions of the 1 Dhc (Fig. 7), we can now identify the position of the 1 motor domain within the I1 structure. EM analysis of axonemes isolated from the E2 and G4 transformants has revealed that lobe 2 of the I1 structure corresponds to the missing 1 motor domain (Fig. 8). Lobe 2 is close to the first radial spoke, in a position that may permit direct signaling between the radial spoke and the 1 Dhc motor domain. We predict that the remaining two lobes of the I1 structure represent the positions of the 1β Dhc motor domain and stem region containing the I1 ICs and LCs respectively. We are currently transforming other I1 mutants with the genes for other I1 subunits to identify the polypeptide components that are located within these structural domains (Perrone et al. 1998; Perrone, C.A., E. O'Toole, and M.E. Porter, work in progress).

The Role of the 1 Dhc Motor Domain in Motility and Phototaxis
The recovery of transformants missing only the 1 Dhc motor domain but containing the other components of the I1 complex has allowed us to analyze the specific role of the 1 Dhc motor domain in flagellar motility. I1 mutants lacking both the 1 and 1β Dhcs have a slow, smooth swimming phenotype with an altered flagellar waveform (Kamiya et al. 1991; Porter et al. 1992). Measurements of swimming velocities reveal that the Dhc1 transformants with truncated 1 Dhcs swim faster than I1 mutants but slower than control strains containing both Dhcs (Table ). The 1 Dhc motor domain, therefore, contributes directly to force production during motility.

The I1 complex is also an essential component of the phototaxis response in Chlamydomonas. Strains that have defects in outer dynein arms, the dynein regulatory complex, or other inner arm isoforms can phototax, but mutants lacking the I1 complex cannot (King and Dutcher 1997). Analysis of other phototaxis mutants that retain the I1 complex reveals that the phosphorylation state of IC138 is altered (King and Dutcher 1997). In vitro sliding assays have shown that the phosphorylation state of the IC138 affects microtubule sliding velocities (Habermacher and Sale 1997). These results suggest a model in which the phosphorylation state of the IC138 modulates the activity of the I1 Dhc motor domains.

To assess the specific role of the 1 Dhc motor domain in phototaxis, we compared the swimming behavior of the Dhc1 transformants to that of control cells in response to a directional light source. pf28 cells were clearly phototactic, but the Dhc1 transformants with truncated 1 Dhcs remained uniformly dispersed during the time course of our assays. These observations indicate that the motor activity of the 1 Dhc contributes to phototaxis, at least in the absence of the outer arms. However, if the outer arms were present, the Dhc1 transformant strains could undergo phototaxis, whereas I1 mutant strains could not (Table ). This difference in behavior in the presence or absence of outer arms suggests that there are cooperative interactions between the I1 complex and the outer arms during the phototaxis response.

Previous studies have demonstrated that differences in the activity of the cis and trans flagellum are the basis of the phototaxis response (Kamiya and Witman 1984; Rüffer and Nultsch 1991, Rüffer and Nultsch 1997; Horst and Witman 1993; Witman 1993). This differential activity includes both differences in beat frequency between the two flagella as well as differences in flagellar waveforms (Sakakibara and Kamiya 1989; Rüffer and Nultsch 1991). The outer dynein arms are responsible for generating the differences in beat frequency observed between cis and trans flagella, as the cis-trans frequency differential is lost in mutants that lack the outer arm or the outer arm Dhc (Sakakibara and Kamiya 1989; Sakakibara et al. 1991). More recent work has demonstrated that this differential beat frequency depends on the presence of the docking structure that facilitates the attachment of the outer arm to its specific binding site on the A-tubule (Takada and Kamiya 1997). The differential in beat frequency is not essential for the phototaxis response (Rüffer and Nultsch 1991), as outer arm mutant cells such as pf28 are capable of phototaxis (Table ). However, it is clear that the cis-trans differences in beat frequency and flagellar waveform must be coupled in some way, because phototaxis mutants such as ptx1 are defective in both (Rüffer and Nultsch 1997; Takada and Kamiya 1997). In this context, it appears that regulation of the I1 complex is important for generating the asymmetries in flagellar waveform between the cis and trans flagella that contribute to phototaxis (King and Dutcher 1997), whereas regulation of the outer arm contributes to the differential in flagellar beat frequency (Takada and Kamiya 1997). Phototaxis can occur in the absence of the outer arms, but not the I1 complex (King and Dutcher 1997; Table ). However, if both the outer arms and part of the I1 complex are present (such as in G4+OA or E2+OA), then their combined activity can apparently compensate for the absence of the 1 Dhc motor domain.

These observations raise several interesting questions about the mechanism by which changes in the phosphorylation state of IC138 might contribute to phototaxis. For example, where is IC138 located in the axoneme relative to the motor domains of the two I1 Dhcs and the three outer arm Dhcs? Is it in lobe 3 of the I1 structure, which is also in close proximity to at least one outer arm per axoneme repeat? Does IC138 interact directly with either the 1 or 1β Dhc? We are planning to address these questions by analyzing subunit interactions within the I1 complex. Other important questions concern the identity and location of the axonemal kinases and phosphatases that modulate the phosphorylation state of IC138. Work from other laboratories has indicated that many of these regulatory components are tightly bound to the flagellar axoneme (Habermacher and Sale 1997; King and Dutcher 1997; Yang, P., and W.S. Sale. 1998. The M_r 140,000 intermediate chain of Chlamydomonas flagellar inner arm dynein is a WD-repeat containing protein implicated in dynein arm anchoring. Mol. Biol. Cell. 9:3335–3349), but the specific enzymes that act on either the outer arm or the I1 IC138 in situ have not yet been identified. The future identification and localization of these regulatory components will provide new insights into the pathway that governs the activity of the multiple dynein motors during flagellar motility and phototaxis.

	Acknowledgments

 
We wish to thank other members of the Porter laboratory for their advice and support during the course of this project. We are also grateful to the members of the laboratories of C. Silflow, P. Lefebvre, and R. Linck (University of Minnesota) for their helpful suggestions at our weekly group meetings. Parts of this work were completed by S.H. Myster in partial fulfillment of the requirements for a Ph.D. degree at the University of Minnesota.

This work was supported by a grant from the National Institutes of General Medical Sciences (GM 55667) to M.E. Porter and S.H. Myster was supported in part by a research training grant from the National Science Foundation for Interdisciplinary Studies on the Cytoskeleton (DIR91134444). E. O'Toole was supported by a National Institutes of Health Biotechnology Resource (RR00592) grant to J.R. McIntosh.

Submitted: 27 May 1999

Revised: 19 July 1999

Accepted: 20 July 1999

used in this report: BAC, bacterial artificial chromosome; Dhc, dynein heavy chain; IC, intermediate chain; LC, light chain; OA, outer arm; P-loop, phosphate binding motif; P1-P4, Pn, Pc, P-loops 1-4, NH2- and COOH-terminal P-loop; RT-PCR, reverse transcriptase PCR

	References

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